The long-term objective of this proposal is to elucidate the mechanism(s) by which polar lipids are flip-flopped across biogenic (self-synthesizing) membranes such as the endoplasmic reticulum (ER). This is an important, unresolved question in membrane biology. Discovering the molecular basis of flipping is essential to understanding how the phospholipid bilayer of biomembranes is propagated, and how the topologically complex syntheses of various glycolipids are executed. The latter processes are required for assembling biologically important cell surface molecules such as N-glycosylated and glycosylphosphatidylinositol (GPI)-anchored proteins in eukaryotes, and O-antigen-modified lipopolysaccharide in Gram-negative bacteria. (Glyco)phospholipid flip-flop does not occur at an appreciable rate in protein-free liposomes but occurs rapidly in the ER via a protein-dependent, metabolic energy-independent, bi-directional, facilitated diffusion process. We hypothesize that specific proteins, biogenic membrane flippases, facilitate the transbilayer diffusion of polar lipids in the ER, including the glycerophospholipids and isoprenoid-based glycolipids that are the focus of this proposal. The characteristics of lipid flip-flop in the ER rule out the participation of ABC transporters that have been identified as potential lipid translocators in other membrane settings. No biogenic membrane flippases have been identified that flip glycerophospholipids and isoprenoid-P-sugars, but compelling genetic evidence points to a membrane protein, Rft1p, as a flippase for dolichol-pyrophosphate based glycolipids in the ER-localized pathway of protein , N-glycosylation. We propose 2 specific aims: to characterize and identify a glycerophospholipid flippase from yeast and rat liver ER and to test biochemically the role of yeast Rft1p in flipping dolichol-PP-based glycolipids. These studies will make use of procedures that we have developed for the functional reconstitution and assay of lipid flip-flop in proteoliposomes generated from a detergent-extract of ER. We anticipate that our results will define a new class of membrane proteins for which no clear prototype currently exists and begin to address our eventual goal of obtaining a molecular definition of the mechanism of lipid flip-flop in biogenic membranes.